To enable autophagy studies, we offer specific tools to report LC3B localization: an antibody against LC3B, and Premo™ sensors. The Premo™ Autophagy Sensors are genetically encoded constructs expressed as either GFP or RFP fused to the N-terminus of LC3B, with the C-terminus free for cleavage and subsequent recruitment of autophagosomes.
Imaging Autophagy in Physiologically Relevant Models
Figure 1. Expression of the Premo™ Autophagy Sensor LC3B-GFP in primary cell types. Rat hippocampal neurons (A), human dermal fibroblasts (B), and human skeletal muscle cells (C) were transduced with the Premo™ Autophagy Sensor LC3B-GFP. This autophagy sensor can be easily combined with other fluorophores to obtain a multiparametric readout of autophagy. In B, the fibroblasts were also transduced with CellLight® Mitochondria-RFP and stained with Hoechst 33342 and Alexa Fluor® 647 phalloidin to label mitochondria (red), nuclei (magenta), and filamentous actin (cyan), respectively. In C, the cells were transduced with CellLight® Mitochondria-RFP and stained with Hoechst 33342 (blue).
Involvement of Other Cellular Organelles
To visualize the formation of autolysosomes, it may be beneficial to use the Premo™ Autophagy Sensor LC3B-RFP rather than LC3B-GFP, as the pKa of TagRFP is lower than that of GFP. This lower pKa renders the TagRFP-based sensor more suitable for visualizing autolysosome formation, as its fluorescence is less likely than that of emGFP to be quenched by the acidic environment of the autolysosomes .
Figure 2. Multicolor imaging of autophagosomes and other organelles. A549 cells were transduced with the Premo™ Autophagy Sensor LC3B-GFP and CellLight® reagents to label specific organelles. At 24 hr after transduction, cells were treated with 50 µM chloroquine overnight to favor autophagosome formation. Nuclei were labeled with Hoechst 33342 (blue). Cells were transduced with (A) CellLight® Mitochondria-RFP, (B) CellLight® Golgi-RFP, (C) CellLight® Lysosomes-GFP, and (D) CellLight® ER-RFP.
Tracking Autophagy From Start to Finish
- Klionsky DJ (2007) Nat Rev Mol Cell Biol 8:931–937.
- Mizushima N, Levine B, Cuervo AM et al. (2008) Nature 451:1069–1075.
- Rubinsztein DC, Gestwicki JE, Murphy LO et al. (2007) Nat Rev Drug Discov 6:304–312.
- Mizushima N, Yoshimori T, Levine B (2010) Cell 140:313–326.
- Hailey DW, Rambold AS, Satpute-Krishnan P et al. (2010) Cell 141:656–667.
- Yu L, McPhee CK, Zheng L et al. (2010) Nature 465:942–946.
- Saftig P, Klumperman J (2009) Nat Rev Mol Cell Biol 9:623–635.
- Kimura S, Noda T, Yoshimori T (2007) Autophagy 3:452–460.
Find more information on LC3B protein detection.
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